CN104357369A - Biological control streptomyces sp. and application thereof - Google Patents
Biological control streptomyces sp. and application thereof Download PDFInfo
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- CN104357369A CN104357369A CN201410691633.1A CN201410691633A CN104357369A CN 104357369 A CN104357369 A CN 104357369A CN 201410691633 A CN201410691633 A CN 201410691633A CN 104357369 A CN104357369 A CN 104357369A
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- lnu
- streptomycete
- streptomyces
- fusarium
- biological control
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention relates to biological control streptomyces sp. and an application thereof. The biological control streptomyces sp. is called as streptomyces sp. Lnu-12; the preservation number is CCTCC NO:M2014554. By virtue of application of a flat confrontation method, the antagonistic action of Lnu-12 on a plurality of plant pathogenic bacteria is detected; Lnu-12 has highest inhibition action on melon fusarium, and the average width of bacteriostatic bands reaches 19-21mm; the Lnu-12 has a high inhibition action on fulvia fulva and alternaria solani, and the widths of bacteriostatic bands are 15mm and 17mm respectively; the Lnu-12 has a certain inhibition action on capsicum root rot fungus, and the width of the bacteriostatic band is 13mm. The streptomyces sp. Lnu-12 has good application prospects in biological control of soil-borne diseases of vegetables in greenhouses.
Description
Technical field
The present invention relates to microbial technology field, particularly a kind of streptomycete bacterial strain and application thereof with the anti-plurality of plant diseases pathogenic bacteria of efficient, wide spectrum.
Biological control streptomycete alleged by the present invention, called after streptomycete Lnu-12 (Streptomyces sp.lnu-12), on November 6th, 2014, in China typical culture collection center preservation, deposit number is CCTCC NO:M2014554.
Background technology
Utilize the method for Agro-chemicals control insect, it is the most popular method of current farm crop pest control, but the environmental pollution caused by excessive use of chemical pesticide and person poultry safety's problem have become the whole world shows great attention to focus, it is human kind sustainable development implementation problem demanding prompt solution.In recent years, meet environmental protection, health, sustainable development theory biological pesticide exploitation become the Main Means solving an above-mentioned difficult problem, biological control, as important measures of control of plant disease, has caused the great attention of people.
Summary of the invention
The object of the present invention is to provide a kind of environmental protection, the biological control streptomycete of the anti-plurality of plant diseases pathogenic bacteria of efficient, wide spectrum.
Another object of the present invention is to provide the application of streptomycete Lnu-12 in Suppressing phytopathogens.
Biological control streptomycete of the present invention, called after streptomycete Lnu-12 (Streptomyces sp.lnu-12), on November 6th, 2014, in Wuhan China typical culture collection center preservation, deposit number is CCTCC NO:M2014554.
Streptomycete Lnu-12 of the present invention is separation screening and obtaining from the green house of vegetables soil of Dongling District, Shenyang City.By pedotheque dilution 10
5, utilize Gause I substratum, adopt conventional plate streaking partition method to carry out separation and Culture, the single bacterium colony of picking actinomycetes, numbers for subsequent use, screens and obtain.
1, streptomycete Lnu-12 morphological specificity:
Lnu-12 is well-grown on Gause I substratum, and Fig. 1 is the gentle raw hypha form of the fibrillae of spores of Lnu-12 under opticmicroscope 100X, and aerial hyphae is flourishing, multi-branched; Fibrillae of spores mostly is straight or curve shape, without closing twist; Spore is rounded, oval.Fig. 2 is Lnu-12 cultural characteristic (front) on Gause I substratum, and Fig. 3 is Lnu-12 cultural characteristic (reverse side) on Gause I substratum, and Fig. 2 and 3 is presented at colony edge without water suction spot, produces without soluble pigment.All meet the feature of streptomyces.
2, Lnu-12 physio-biochemical characteristics:
Gelatine liquefication, hydrogen peroxide experiment, cellulose hydrolysis, lecithinase experiment are all positive; Milk solidify with peptonize, Starch Hydrolysis, MR test, hydrogen sulfide is tested, tryptophane decomposition experiment, nitrate reduction are tested and be feminine gender; Glucose, fructose, wood sugar, lactose, starch, sucrose, semi-lactosi, cellobiose can be utilized as carbon source for growth, and biochemical reactions characteristic is consistent with starch chain mould.
3, the 16S rDNA sequence analysis method of streptomycete Lnu-12:
(1) DNA extraction of streptomycete Lnu-12 adopts multigelation method, carries out pcr amplification as template.
(2) PCR primer: forward primer BSF8/27:5 '-AGAGTTTGATCCTGGCTCAG-3 ';
Reverse primer BSR1541/1522:5 '-AAGGAGGTGATCCAGCCGCA-3 '.
(3) PCR primer is synthesized by Shanghai biotechnology company limited.
(5) pcr amplification condition: 94 DEG C of 5min; 94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 1.5min; 30 circulations; 72 DEG C of 5min.
Streptomycete Lnu-12 bacterial strain of the present invention, PCR primer order-checking is completed by Shanghai biotechnology company limited, the sequence recorded is as shown in SCQ ID No.1, measured 16S rDNA sequence blast search instrument is recalled relative 16S rDNA sequence from GeneBank, then Multiple Sequence Alignment is carried out with CLASTAL X software, and adopting ortho position phase connection (Neighbor-Joining) in MEGA4.1 software to carry out structure and the tetraploid rice of systematic evolution tree, constructed systematic evolution tree is shown in Fig. 4.
Streptomycete Lnu-12 bacterial strain is through round pcr, DNA sequencer is analyzed, its 16S rDNA sequence 1424bp, the 16S rDNA sequence of related strain in itself and GeneBank database compared, result reaches 99% with starch chain mould dark grey subspecies (S.diastaticus subsp.Ardesiacus) homology reported.
The present invention is separated and obtains biocontrol strain Lnu-12 from the green house of vegetables soil of Dongling District, Shenyang, pass through biochemical reactions, and utilize PCR to react the 16S rDNA of this bacterial strain of amplification, through order-checking qualification, CLASTAL X software is utilized to carry out Multiple Sequence Alignment, then use ortho position phase connection (Neighbor-Joining) in MEGA4.1 software to carry out the structure of systematic evolution tree, qualification streptomycete Lnu-12 (Streptomyces sp.lnu-12) bacterial strain is starch chain mould dark grey subspecies (S.diastaticus subsp.Ardesiacus).
The invention has the beneficial effects as follows: streptomycete Lnu-12 provided by the present invention has antagonistic action to various plants pathogenic bacteria, wherein the strongest to Fusarium oxysporum restraining effect, antibacterial band width average reaches 19 ~ 21mm.Streptomycete Lnu-12 provided by the present invention has the antimicrobial spectrum of high-efficiency broad spectrum, and anti-Plant diseases ability is stronger.For leaf muld of tomato, carried out field efficacy experiment with Lnu-12 fermented liquid, have significant prevention effect to leaf muld of tomato after result display Lnu-12 fermented liquid dilutes 50 times, prevention effect can reach 70%, apparently higher than contrast medicament.
Accompanying drawing explanation
Streptomycete Lnu-12 (Streptomyces sp.lnu-12), depositary institution: China typical culture collection center, is called for short: CCTCC, depositary institution address: China, Wuhan University, postcode: 430072.Preservation date is on November 6th, 2014, and deposit number is CCTCC NO:M2014554.
Fig. 1 is the gentle raw hypha form of the fibrillae of spores of Lnu-12 (opticmicroscope 100X).
Fig. 2 is Lnu-12 cultural characteristic (front) on Gause I substratum.
Fig. 3 is Lnu-12 cultural characteristic (reverse side) on Gause I substratum.
Fig. 4 is the phylogenetic evolution tree of Lnu-12 bacterial strain.
Embodiment
The bacteriostatic action of embodiment 1 streptomycete Lnu-12 (Streptomyces sp.lnu-12)
1, activate: by streptomycete Lnu-12 (Streptomyces sp.lnu-12) (CCTCC NO:M 2014554), aseptic operating platform operates, be evenly coated on Gao Shi No. 1 inclined-plane solid plate pick the bacterium liquid of preservation with aseptic inoculation ring after, cultivate 3 ~ 5 days for 28 DEG C.
2, adopt dull and stereotyped face-off method, scraping, through the Lnu-12 spore prepare suspension of overactivation, is evenly coated on Gao Shi No. 1 substratum solid plate, cultivates 3 ~ 5 days for 28 DEG C.
3, make bacterium cake with the aseptic punch tool of diameter 7mm, be positioned over the dull and stereotyped central authorities of PDA, cultivate after 2 days for 28 DEG C, then to keep to the side six kinds of various pathogenic bacteria bacterium cakes that equidistant inoculation same method makes in surrounding, cultivate 4 ~ 6 days for 28 DEG C, the antibacterial situation of observed and recorded.Often process repetition 3 times.
Six kinds of different pathogenic bacterias are: Fusarium Solani (Pepper Root Rot), Fulvia fulva (Cladosporium fulvum), tomato early blight bacterium (Alternaria solani), Melon fusarium Wilt (Fusariwn oxysporum), withered germ of water-melon (Fusarium oxysporum f.sp.niveum), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum).
4, result is as table 1
Table 1
| Pathogenic bacteria | Antibacterial band width average |
| Fusarium Solani (Pepper Root Rot) | 13mm |
| Fulvia fulva (Cladosporium fulvum) | 15mm |
| Tomato early blight bacterium (Alternaria solani) | 17mm |
| Melon fusarium Wilt (Fusariwn oxysporum) | 19mm |
| Withered germ of water-melon (Fusarium oxysporum f.sp.niveum) | 20mm |
| Cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum) | 21mm |
From table 1, streptomycete Lnu-12 of the present invention all has certain restraining effect to all strains testeds.Wherein the strongest to Fusarium oxysporum restraining effect, reach 19 ~ 21mm, have very strong restraining effect to Fulvia fulva, tomato early blight bacterium, antibacterial bandwidth is respectively 15mm and 17mm, has certain restraining effect to Fusarium Solani, antibacterial bandwidth 13mm.
Embodiment 2 biological control agents Lnu-12 fermented liquid is to the prevention effect of leaf muld of tomato
By bacterial strain Lnu-12 28 DEG C of cultivations on Gause I slant medium that the present invention obtains, make spore suspension after producing enough spores, the amount with 3% is inoculated in fermention medium (Semen Maydis powder 4.0%, KNO in 250ml triangular flask
30.2%, FeSO
40.02%, NaCl 0.02%, CaCO
30.2%, surplus is water) in, 28 DEG C, 180r/min shaking culture 5 days.The centrifugal 20min of fermented liquid 7000r/min, supernatant liquor is put in 4 DEG C of refrigerators for subsequent use.
The mode of potted plant inoculation is adopted in glasshouse, by Fulvia fulva 26 DEG C of constant temperature culture 4-5d on PDA substratum, get 7-8 leaf phase tomato seedling No. 5 syringe needles and sting 4 pins at middle part blade, then Fulvia fulva bacterium cake is attached to acupuncture place, dip in sterilized water with sterile absorbent cotton and cover moisturizing on it, often process 15 strains, repeat for 3 times, 28-30 DEG C of high-temperature cultivation, removes bacterium cake after moisturizing 48h.By above-mentioned fermented supernatant fluid 50 times of diluents, 50% carbendazim WP 500 times liquid and clear water contrast are evenly sprayed on the face anyway of blade, drip for degree with blade face.Inoculate 7 days " Invest, Then Investigate " disease indexs, calculate prevention effect.
Present invention employs antifungal medicine derosal conventional in production is positive control, potted plant experiment result shows, adopt the fermentation condition of above-mentioned optimization, the fermented liquid of bacterial strain Lnu-12 has good drug effect to leaf muld of tomato, after process, disease index is 20.5, be starkly lower than contrast, prevention effect can reach 69.9%.Be better than contrast medicament 50% carbendazim WP.
Table 2Lnu-12 fermented liquid is to the prevention effect of leaf muld of tomato
Claims (4)
1. a biological control streptomycete, is characterized in that: called after streptomycete Lnu-12 (Streptomyces sp.lnu-12), and in China typical culture collection center preservation, deposit number is CCTCC NO:M 2014554.
2. the application of streptomycete Lnu-12 according to claim 1 in Suppressing phytopathogens.
3. apply as claimed in claim 2, it is characterized in that method is as follows: streptomycete Lnu-12 is fermented in fermention medium, after fermented supernatant fluid being diluted 50 times, be applied in the mode of spraying and suffer from the plant leaf of phytopathogen.
4. apply as claimed in claim 2 or claim 3, it is characterized in that: described phytopathogen is: Fusarium Solani (Pepper Root Rot), Fulvia fulva (Cladosporium fulvum), tomato early blight bacterium (Alternaria solani), Melon fusarium Wilt (Fusariwn oxysporum), withered germ of water-melon (Fusarium oxysporum f.sp.niveum), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410691633.1A CN104357369B (en) | 2014-11-25 | 2014-11-25 | Biological control streptomycete and its application |
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|---|---|---|---|
| CN201410691633.1A CN104357369B (en) | 2014-11-25 | 2014-11-25 | Biological control streptomycete and its application |
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| Publication Number | Publication Date |
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| CN104357369A true CN104357369A (en) | 2015-02-18 |
| CN104357369B CN104357369B (en) | 2017-10-13 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061551A (en) * | 2019-11-27 | 2021-07-02 | 吉林省林业科学研究院 | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria |
| CN114410518A (en) * | 2022-01-07 | 2022-04-29 | 春华秋实科技集团有限公司 | Compound microbial agent containing streptomyces and application thereof |
| CN116083266A (en) * | 2022-07-28 | 2023-05-09 | 西北农林科技大学 | Streptomyces flagelliformis, biocontrol microbial inoculum and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182483A (en) * | 2007-10-31 | 2008-05-21 | 浙江工业大学 | Streptomycete S1-5 and uses thereof |
| CN103468599A (en) * | 2013-05-22 | 2013-12-25 | 滨州学院 | Streptomycetes sp. and its application in winter jujube ring rot prevention and treatment |
-
2014
- 2014-11-25 CN CN201410691633.1A patent/CN104357369B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182483A (en) * | 2007-10-31 | 2008-05-21 | 浙江工业大学 | Streptomycete S1-5 and uses thereof |
| CN103468599A (en) * | 2013-05-22 | 2013-12-25 | 滨州学院 | Streptomycetes sp. and its application in winter jujube ring rot prevention and treatment |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061551A (en) * | 2019-11-27 | 2021-07-02 | 吉林省林业科学研究院 | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria |
| CN114410518A (en) * | 2022-01-07 | 2022-04-29 | 春华秋实科技集团有限公司 | Compound microbial agent containing streptomyces and application thereof |
| CN116083266A (en) * | 2022-07-28 | 2023-05-09 | 西北农林科技大学 | Streptomyces flagelliformis, biocontrol microbial inoculum and application |
| CN116083266B (en) * | 2022-07-28 | 2024-05-03 | 西北农林科技大学 | Streptomyces flagelliformis, biocontrol microbial inoculum and application |
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| CN104357369B (en) | 2017-10-13 |
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